Tài liệu Báo cáo khoa học: Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani pdf

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Tài liệu Báo cáo khoa học: Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani pdf

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Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani He ´ ctor Villa 1 , Yolanda Pe ´ rez-Pertejo 1 , Carlos Garcı ´ a-Estrada 1 , Rosa M. Reguera 1 , Jose ´ Marı ´ a Requena 2 , Babu L. Tekwani 3 , Rafael Balan ˜ a-Fouce 1 and David Ordo ´ n ˜ ez 1 1 Departamento de Farmacologı ´ a y Toxicologı ´ a (INTOXCAL), Facultad de Veterinaria, Universidad de Leo ´ n, Spain; 2 Centro de Biologı ´ a Molecular ‘Severo Ochoa’, Universidad Auto ´ noma de Madrid, Spain; 3 National Center for Natural Products Research, School of Pharmacy, University of Mississippi, USA ATP-regenerating enzymes may have an important role in maintaining ATP levels in mitochondria-like kinetoplast organelle and glycosomes in parasitic protozoa. Adenylate kinase (AK) (ATP:AMP phosphotransferase) catalyses the reversible transfer of the c-phosphate group from ATP to AMP, releasing two molecules of ADP. This study describes cloning and functional characterization of the gene encoding AK2 from a genomiclibrary of Leishmania donovani and also its expression in leishmania promastigote cultures. AK2 was localized on an 1.9-Mb chromosomal bandas a single copy gene. L. donovani AK2 gene is expressed as a single 1.9-kb mRNA transcript that is developmentally regulated and accumulated during the early log phase. The overexpression of L. donovani AK gene in Escherichia coli yielded a 26-kDa polypeptide that could be refolded to a functional protein with AK activity. The recombinant protein was purified to apparent homogeneity. Kinetic analysis of purified L. donovani AK showed hyperbolic behaviour for both ATP and AMP, with K m values of 104 and 74 l M , respectively. The maximum enzyme activity (V max ) was 0.18 lmolÆmin )1 Æ mg )1 protein. P 1 ,P 5 -(bis adenosine)-5¢-pentaphosphate (Ap 5 A), the specific inhibitor of AK, competitively inhibited activity of the recombinant enzymes with estimated K i values of 190 n M and 160 n M for ATP and AMP, respectively. Ap 5 A also inhibited the growth ofL. donovani promastigotes in vitro which could be only partially reversed by the addition of ADP. Thus, presence of a highly regulated AK2, which may have role in maintenance of ADP/ATP levels in L. donovani, has been demonstrated. Keywords: adenylate kinase; functional expression; leish- mania. Adenylate kinase (AK) (ATP:AMP phosphotransferase, EC 2.7.4.3) catalyses the reversible transfer of the c-phos- phate group from ATP to AMP, releasing two molecules of ADP [1]. Different isoenzymic forms of AK are involved in maintenance of constant intracellular levels of adenine nucleotides, necessary for energy metabolism and nucleic acid synthesis [2]. Three AK isoforms have been described in mammals: AK1 in the cytosol, AK2 in the mitochondrial intermembrane space [3] and AK3, a GTP:AMP phospho- transferase that resides exclusively in the mitochondrial matrix [4]. The role of AKs in the organisms of the order Kineto- plastida (that includes trypanosomes, leishmanias and other pathogenic parasites) has not been studied in detail yet. As in their mammalian hosts, AK in these parasites seems to be distributed in several intracellular compartments. These eukaryotic microorganisms have some characteristic sub- cellular organelles, such as modified mitochondria called ÔkinetoplastsÕ and several specific energy-producing micro- bodies called ÔglycosomesÕ [5]. AK plays an important role in the ATP-regenerating system required for eukaryotic ciliary or flagellar movements and has been found to be associated with Tetrahymena cilia [6], Paramecium caudatum [7] as well as vertebrate spermatozoid flagella [8,9]. AK activity in Leishmania promastigotes and Trypanosoma spp. has been found to be associated with the membrane of glycosomes [10,11]. A third form of AK located in the cytosol has been proposed as a virulence factor in bacteria, as it is secreted along with other ATP-related enzymes, contributing to modulation of ATP levels during macrophage death [12,13]. Structural studies with AKs from different sources have revealed the presence of three distinct domains: the rigid CORE-domain and two smaller peripheral domains or mobile parts, the NMP-binding site and the LID-domain [4]. The NMP-binding site has many intermolecular contacts with the nucleotide phosphoryl acceptor (NMP), whereas the LID-domain prevents the hydrolysis of the Mg-bound phosphoryl donor in the active site. The relative movement of the NMP-binding site and LID-domain Correspondence to D. Ordo ´ n ˜ ez, Department Farmacologı ´ ayToxi- cologı ´ a (INTOXCAL), Ftad. Veterinaria, Universidad de Leo ´ n, Campus de Vegazana s/n 24071 Leo ´ n, Spain. Fax: + 34 987 291 252, Tel.: + 34 987 291 590, E-mail: dftrbf@isidoro.unileon.es Abbreviations: AK, adenylate kinase; IPTG, isopropyl thio-b- D - galactoside; Ap 5 A, P 1 ,P 5 -bis(adenosine)-5¢-pentaphosphate; NMP, nucleoside monophosphate; IC 50 , 50% inhibitory concentration. Enzymes: adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3). Note: The nucleotide sequence data reported has been submitted to EMBL and GenBank Nucleotide Sequence Databases under the accession number AF156853. (Received 14 May 2003, revised 25 July 2003, accepted 9 September 2003) Eur. J. Biochem. 270, 4339–4347 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03826.x induced by the substrates leads to two conformations called ÔclosedÕ and ÔopenÕ states, which have been thoroughly described by Schulz et al. [14]. Genes encoding AKs have been characterized in many organisms, including bacteria, fungi and mammals. There are no reports about character- ization of this enzyme in parasitic protozoa. Leishmania donovani is the aetiological agent for visceral leishmaniasis, a devastating disease which is mostly endemic in Asian and Mediterranean countries [15]. This paper describes the cloning and functional characterization of an AK gene from a genomic library of L. donovani as well as its expression and molecular characterization. Materials and methods Materials Plasmids pGEM-3Zf(+) and pQE30 were from Promega and QIAGEN, respectively. [ 32 P]dCTP[aP] (3000 CiÆ mmol )1 ) was from DuPont-NEN. Restriction enzymes and Taq DNA polymerase were from Boehringer Mann- heim. Isopropyl thio-b- D -galactoside (IPTG), pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, pro- teinase K, antibiotics, ATP, AMP, dithiothreitol and P 1 ,P 5 - bis(adenosine)-5¢-pentaphosphate (Ap 5 A) were from Sigma. Lambda EMBL-3 genomic library was a gift from J.C. Meade (University of Mississippi, Medical Center, MS, USA). Parasite cell culture Promastigote cultures of L. donovani 1S2D strain (a gener- ous gift from L. Rivas, Centro de Investigaciones Biolo ´ gicas CSIC, Madrid, Spain) were grown at 26.5 °C in M199 medium (Sigma) supplemented with sodium bicarbonate, 5m M Hepes, heat inactivated foetal bovine serum (10%) (Boehringer Mannheim) and gentamicin (30 lgÆmL )1 ) (Sigma). Nucleic acid isolation Genomic DNA from L. donovani promastigotes was isola- ted from2.0 · 10 9 cells in 10 m M EDTA, 150 m M NaCl, 0.4% SDS, 50 mgÆmL )1 proteinase K and incubated at 65 °C for 1 h followed by an overnight incubation at 37 °C. The DNA was further purified by phenol/chloroform extraction and ethanol precipitation. RNA was isolated with RNAeasy kit (QIAGEN) according to manufacture’s protocol. Isolation of a L. donovani AK gene fragment by PCR To generate a DNA probe for the isolation of the L. donovani AK gene, PCR was carried out using degenerate oligonucleotides based on conserved amino acid sequences in human AK type 2A [16], Saccharomyces cerevisiae [17], Trichomonas vaginalis [18], Oryza sativa [19] and Escherichia coli [20] AK proteins. The sense primer (5¢-GACGG TTTTCCGCGCAC-3¢) corresponding to the amino acid residues DGFPRT and an antisense primer (5¢-ACCAGG GGCTCGCCGGT-3¢) corresponding to amino acid resi- dues TGEPLV were used for isolation of AK gene fragment by PCR with L. donovani genomic DNA. The PCR product was sequenced on both strands by an ALF automated sequencer (Sistemas Geno ´ micos, Valencia, Spain) according to the dideoxy chain termination method using fluorescent primers and T7 DNA polymerase. Isolation of L. donovani AK gene The EMBL L. donovani library was used for screening. 50 000 bacteriophages were blotted onto positively charged nylon membranes (Amersham) and prehybridized for 4 h at 42 °Cin5· Denhardt’s solution, 5 · NaCl/Cit, 50 m M sodium phosphate (pH 6.5), 100 lgÆmL )1 salmon sperm DNA, and 50% formamide. Membranes were hybridized for 16–20 h at 42 °C in the same buffer containing 10 6 )10 7 c.p.m. of 32 P-labelled 215 bp-PCR product, that had been radiolabelled by the random primer method with [ 32 P]dCTP[aP] (Amersham). Filters were washed twice with 2 · NaCl/Cit plus 0.1% SDS at 42 °C for 10 min each, followed by two washes in 1 · NaCl/Cit, plus 0.1% SDS at 42 °C for 10 min. Positive bacteriophages were carried through secondary and tertiary screenings, until all the plaques on the plate appeared positive. The DNA was isolated from amplified phages by liquid culture method as described by Sambrook et al. [21]. Subcloning and sequencing of L. donovani AK gene The isolated bacteriophage DNA was digested with restric- tion endonucleases, separated by electrophoresis on 0.7% agarose gels, and transferred to positively charged nylon membranes by the method of Southern [22]. Southern blots were probed with the 215-bp PCR product under the same conditions described above for the screening of the genomic library. A single 1.6-kb EcoRI–SphI fragment that hybrid- ized to the probe was ligated into pGEM-3Zf(+) and transformed into E. coli DH5a. Large-scale plasmid pre- paration of pGEM-3Zf(+) containing the 1.6-kb EcoRI– SphI fragment was processed using QIAGEN columns, and the insert was sequenced as above. Analyses of nucleotide and amino acid sequences were performed using the BLAST algorithm from the database National Center for Biotech- nology Information. Southern and Northern analyses Genomic DNA prepared from L. donovani promastigotes harvested at late log phase was digested with restriction enzymes, as described above, separated by electrophoresis in 0.8% agarose gels and transferred to nylon membranes. Total RNA was extracted from L. donovani promastigotes using RNeasy Mini kit (QIAGEN), following the manu- facturer’s instructions. Approximately 20 lgtotalRNA were resolved onto Mops/formaldehyde 1.2% agarose gels, transferred to a nylon membrane, and fixed to the filters with an UV-crosslinker. The filters were prehybridized for 2 h at 42 °Cin10mL10· Denhardt’s, 6 · NaCl/Cit and 1% SDS solution containing 300 lgÆmL )1 herring sperm DNA. Hybridization was performed overnight at 42 °Cin 10 mL 50% formamide, 6 · NaCl/Cit, 1% SDS, 150 lgÆmL )1 herring sperm DNA and the 32 P-labelled probe. The filters were washed stepwise in 6 · NaCl/Cit 1% 4340 H. Villa et al. (Eur. J. Biochem. 270) Ó FEBS 2003 SDS for 30 min at room temperature, 1 · NaCl/Cit, 0.5% SDS for 45 min at 42 °Cand0.1· NaCl/Cit 0.2% SDS for 45 min at the same temperature. Finally the filters were exposed at )70 °C for autoradiography. Chromosomal localization of AK gene Promastigotes (2 · 10 8 ) were harvested by centrifugation, washed twice in NaCl/P i and resuspended in NaCl/P i mixed 1 : 1 with 2% agarose. Processing of the samples was carried out at 50 °Cfor48 hin10 mL0.5 M EDTA pH 8.0, 1% Sarkosyl, and 150 lLof2mgÆmL )1 freshly prepared proteinase K. Separation of the chromosomal bands was achieved by electrophoresis at 14 °Cin1%agarosegels with 0.5 · TBE running buffer, using a Clamped Homo- geneous Electrical Field apparatus (CHEF, BIO-RAD) with a 35–120 s ramping pulse at 6 VÆcm )1 for 33 h. S. cerevisiae (Amersham) chromosomes were used as molecular weight markers. After staining with ethidium bromide, gels were blotted onto nylon filters (Sigma) by alkaline transfer. The membrane was probed to the 690-bp probe as described above. Heterologous expression The AK gene was amplified by PCR from L. donovani genomic DNA. The sense primer 5¢-ACATGCATGCAT GAAGATCGTGATGGAAGG-3¢ introduces an SphI restriction site and the antisense one 5¢-AACTGCAG GCTTTCACCAGAATTTCCACC-3¢ introduces a PstI restriction site to subclone the AK gene in the pGEM- 3Zf(+) cloning vector (Promega), creating pGEM-AK. To subclone the AK gene into pQE-30 expression vector (QIAGEN), pGEM-AK was digested with SphIandPstI and the agarose purified AK gene insert was ligated into SphI–PstI digested pQE30 plasmid with T4 DNA ligase (Promega). This construct, called pQE30-AK, was trans- formed into XL1-Blue E. coli competent cells. Overnight cultures prepared from single colonies were used to inoculate 100 mL of Luria–Bertani medium plus ampicilin (50 lgÆmL )1 ). Cells were grown to D 600 0.5–0.8 and IPTG was added to a final concentration of 0.1 m M .After induction with IPTG, growth was continued for 5 h under the same conditions. Cells were harvested by centrifugation, washed in 1 · NaCl/P i and resuspended into 50 m M Tris/ HCl(pH8),10m M MgCl 2 before sonication. Inclusion bodies were obtained by centrifugation at 10 000 g for 10 min at 4 °C. Pellets were washed twice with 4 M urea, 20% Triton-X100, 50 m M Tris/HCl pH 8, 10 m M MgCl 2 , 1m M dithiothreitol and twice with 50 m M Tris/HCl (pH 8), 10 m M MgCl 2 ,1m M dithiothreitol. After incubation with 50 lgÆmL )1 DNAse I (Roche), cell debris were removed by centrifugation and the insoluble fraction was dissolved in equilibration buffer (10 m M Tris/HCl, 300 m M NaCl con- taining 8 M urea) to be loaded onto a nickel column equilibrated with the same buffer. The column was washed stepwise with equilibration buffer and equilibration buffer plus 10 m M imidazole. The elution of AK protein was carried out with equilibration buffer containing 200 m M imidazole, collecting 1-mL fractions which were analysed by SDS/PAGE. Fractions enriched in AK were dialysed stepwise, first against 50 m M Tris/HCl, 1 m M EDTA, 10 m M dithiothreitol, containing 1 M urea for 2 h and then against 50 m M Tris/HCl, 1 m M EDTA, 10 m M dithiothre- itol containing 0.25 M urea overnight [23]. SDS/PAGE and Western blotting L. donovani promastigotes were harvested during exponen- tial growth phase (day 3) and washed twice with NaCl/P i . Once they were sonicated and centrifuged at 10 000 g for 20 min, the supernatant was removed. Fifty lg protein were diluted in loading buffer (60 m M Tris/HCl pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 5% glycerol), heated in a boiling water bath for 5 min, and analysed by SDS/PAGE (12% acrylamide, 2.7% bisacrylamide). Proteins were electrotransferred to nylon membranes for 1 h at 25–30 VÆcm )1 , blots were blocked by incubation in 10 m M Tris/HCl pH 7.5, 1 M NaCl, 0.5% Tween 20, 5% nonfat milk powder (w/v) for 1 h at room temperature. Primary, polyclonal antibodies (obtained from rabbit serum ino- culated with purified recombinant leishmanial AK) were added to this buffer and the blot incubated for an 2 h. The blot was washed extensively in 10 m M Tris/HCl pH 7.5, 1 M NaCl, 0.5% Tween 20 and then incubated with an anti- rabbit antibody conjugated to alkaline phosphatase. Anti- bodies were detected using Nitro-Blue-tetrazolium chloride as substrate (Boehringer Mannheim). AK assay Enzyme activity was assayed in the forward direction by the coupled reactions system [24] adding different concentra- tions of ATP and AMP as the substrates. The rate of NADH disappearance was measured spectrophotometri- cally at 340 nm in a coupled enzyme assay to pyruvate kinase and lactate dehydrogenase at 30 °C. The assay mixture contained, in a total volume of 1 mL, 0.1 m M Tris/ HCl (pH 8.0), 120 m M KCl, 10 m M MgCl 2 ,1m M phos- phoenolpyruvate, 0.2 m M NADH, 4.4 U pyruvate kinase (Sigma), 5 U lactate dehydrogenase (Sigma) and several concentrations of ATP (range 0.025–5 m M )andAMP (range 0.010–1.5 m M ). Protein content was estimated by using the method of Bradford [25]. For analysis of enzyme kinetics AK assay was done at varying concentrations of ATP and AMP and the results were analysed by double reciprocal Lineweaver–Burk plot by using SIGMA PLOT . Kinetic parameters (K m and V max ) were computed from these plots. In vitro anti-leishmania assay Anti-leishmanial activity of Ap 5 A the inhibitor of AK was tested on a transgenic cell line of L. donovani promastigotes expressing firefly luciferase. These cells show constant and stable expression of luciferase, which is directly proportional to the number of live promastigotes. The assay was performed in clear-bottomed, 96-well micro plates. Promas- tigote culture (200 lL; 2 · 10 6 cells per well) was exposed to varying concentrations (5–250 l M )ofAp 5 A. The plates were incubated at 26 °C for 72 h and growth of promastigotes was determined by luciferase assay with Steady Glo reagent (Promega). Fifty per cent inhibitory concentrations (IC 50 ) were determined from the inhibition curves. Ó FEBS 2003 L. donovani adenylate kinase (Eur. J. Biochem. 270) 4341 Results Cloning and characterization of the AK gene in L. donovani An L. donovani (1S2D strain) lambda EMBL3 genomic library was screened with a [ 32 P]dCTP[aP]-labelled AK probe obtained by PCR from genomic DNA of L. dono- vani promastigotes. After three rounds of screening, three different clones were isolated. Analysis by digestion with restriction enzymes indicated that all three clones were the same, therefore only one was sequenced in both strands (Sistemas Geno ´ micos, Valencia, Spain). The analysis of the nucleotide sequence revealed the presence of a 690-bp long ORF that encoded a protein of 230 amino acids, with an estimated molecular mass of 26 kDa. This gene was designated the AK gene (see GenBank accession number AF156853). The amino acid sequence deduced from this ORF showed significant homology with AK enzymes from different organisms. Multiple sequence alignment (Fig. 1) of the L. donovani AK protein with other proteins from phylogenetically diverse organisms revealed that the Leishmania AK gene has the conserved motifs involved in NMP binding in a sequence from 30 to 61 amino acids. The amino acids residues that possibly mediate the interactions with AMP are T31, R36 and L59. In position 35 a valine residue replaced the leucine residue, which is conserved in all other sequences, while at position 60 an isoleucine residue was present in place of a highly conserved valine. The decapeptide GPPQGGKTTV (in position 7–16) in L. donovani AK resembles the P-loop related to the ATP- binding site [26]. The sequence containing four cysteine residues C-X 2 -C-X n -C-X 2 -C predicted to be a zinc- binding motif was not found in L. donovani LID sequence, although it was present in P. falciparum AK (AF308612), where it is located in positions 127–158. The LID domain, said to be related to the active site of the enzyme in most AK, starts at position 124 and extends to position 161 ) a total of 38 amino acids. A number of phylogenetically conserved motifs (GFPRT in position 86–90) or amino acids (R121, G132, R133, R169, Y173, Q176, V201) with no defined functions are also found in L. donovani AK. Southern blot analysis (Fig. 2A) was performed on genomic DNA to determine the gene copy number and genomic organization of AK genes in the L. donovani genome. Several enzymes with cleavage sites in the AK coding region were used to digest the DNA, and the blots were probed with the 690-bp coding region. Complete digestion resulted in a single copy within the L. donovani genome, as all enzymes showed only one band, except those which cut once into the gene sequence (BamHI, KpnIandSalI), which exhibited two hybridizing bands. Chromosomal bands from L. donovani 1S2D strain were resolved by pulsed-field gel electrophoresis as described in Materials and methods, transferred to a nylon membrane and hybridized to the random primer-labelled probe with AK coding region to determine the chromosomal location of the AK gene. A hybridization signal was observed in a chromosomal band of  1.9 Mb (Fig. 2B). Expression of the AK gene during growth of leishmania promastigotes in vitro was also investigated. Total RNA was isolated from promastigotes harvested at different growth periods and analysed by Northern blotting (Fig. 3). The promas- tigotes in these cultures entered the logarithmic phase of growth within 1–2 days after subculturing and reach the stationary phase by day 5. Fig. 3B shows a single RNA transcript of  1.9 kb. Transcription of the AK gene was higher during early period (days 1 and 2) of growth, Fig. 1. Multiple amino acid sequence align- ments of L. donovani AK. The predicted amino acid sequences for L. donovani, Trypanosoma brucei, T. vaginalis, P. falciparum, S. cerevisi- ae, and human 2A AKs were aligned using the CLUSTAL X multiple sequence alignment pro- gram. Symbols: Ô*Õ identical or conserved residues in all sequences in the alignment; Ô:Õ conserved substitutions; Ô.Õ semiconserved substitutions. Complete genomic sequences of AK proteins are available from the GenBank, for L. donovani accession number (AF156853), T. brucei (AF047722), T. vaginalis (U07203), P. falciparum (AF308612), S. cerevisiae (Y00413) and human 2A (U39945). 4342 H. Villa et al. (Eur. J. Biochem. 270) Ó FEBS 2003 diminishing to very low levels in the logarithmic and stationary phases (Fig. 3C). Overexpression and purification of AK2 The cultures of bacterial cells (XL1-Blue) transformed with pQE30-AK were induced with IPTG for overexpression of AK2 gene. Marked overexpression of AK2 protein was noticed but the recombinant AK protein accumulated mainly in the inclusion bodies (Fig. 4A). A 26-kDa polypeptide was detected after induction of the culture with IPTG, which matched the molecular mass predicted from the amino acid sequence of AK2 protein. The purification and functional folding of L. donovani AK from the E. coli cell lysates was carried out by washing and dissolving the inclusion bodies in 8 M urea. Refolding was achieved by equilibrium dialysis against 50 m M Tris/HCl pH 8.0, con- taining either MgSO 4 ,KClor10 m M dithiothreitol, extracts were active and contained over 99% of the AK protein. Finally, the recombinant protein was purified to apparent homogeneity by affinity chromatography on Ni–NTA agarose columns. The recombinant protein was analysed for enzyme activity. Western analysis of exponential phase leishmanial extracts were performed with an anti-AK polyclonal antibody obtained from previously immunized rabbits. A single band of 26 kDa was detected, as shown in Fig. 4B. Functional characterization of recombinant AK The recombinant protein purified from the inclusion bodies and refolded in the presence of dithiothreitol, MgSO 4 and KCl showed significant enzyme activity. AK activity was linear in terms of both incubation time (up to 90 min) and protein concentration (0.5–5 lgprotein;data not shown). Maximum enzyme activity (V max )atsteady- state conditions and saturation concentrations of both substrates in the forward reaction, i.e. 2.5 m M ATP and 1.5 m M AMP, was 0.18 lmol ADP formedÆmin )1 Æmg )1 . The enzyme exhibited hyperbolic behaviour with both ATP and AMP. The K m value estimated for ATP was 104 ± 20 l M , while for AMP it was 74 ± 18 l M . Stability of the recombinant leishmania AK was also checked. The recombinant enzyme protein was incubated, as described previously, at 4 °C, 13 °Cand26.5°Cfor10 consecutive days. Activity was measured at different time Fig. 2. Southern blot analysis of wild-type AK loci. (A) Genomic DNA (20 lg) was isolated from L. donovani promastigotes, digested with PvuI, NotI, SalI, KpnI, BamHI, PstI, and resolved on a 0.7% agarose gel,andblottedontonylonmembranes.Blotswerehybridizedtothe 690-bp PCR fragment under high stringency conditions. (B) Chromo- somal location of the AK gene in the L. donovani 1S2D strain. After pulse-field gel electrophoresis of the L. donovani 1S2D promastigotes different sized groups were displayed, similar to other L. donovani strains. The gel was blotted onto nylon membranes by alkaline transfer andhybridizedtorandomlabelled690-bpPCRproduct.Underthese conditions only one band of  1.9 Mb was obtained. Fig. 3. Northern blots showing AK mRNA abundance during promas- tigote culture growth. Total RNA was isolated from cultured pro- mastigotes in logarithmic phase (days 1–2), late logarithmic early stationary phase (days 3–5) and stationary phase (day 6). (A, B) The RNAs (20 lg per lane) were loaded onto agarose gel, separated by electrophoresis and transferred to a nylon membrane. An ethidium bromide stain of the gel is shown (A). The membrane was probed with the coding region of the AK gene (B). (C) Relative abundance of AK mRNA during the growth curve of promastigotes. Ó FEBS 2003 L. donovani adenylate kinase (Eur. J. Biochem. 270) 4343 intervals as described in Fig. 5. A time-dependent reduc- tion in AK activity was noticed at all temperatures studied, although it was more pronounced at 26.5 °Cthan 4 °C. Semi-inactivation times were estimated to be 29, 24 and 12 h at temperatures of 4, 13 and 26.5 °C, respectively. Inhibition of AK by the specific inhibitor Ap5A and its effect on growth of L. donovani promastigotes in vitro The effect of the nucleotide analogue Ap 5 A, was studied on L. donovani recombinant AK under standard assay conditions (Fig. 6). Different concentrations (50–1000 n M ) of Ap5A were added to the assay buffer, which contained 6 lg refolded AK and three concentrations of one of the nucleotides (ATP: 0.62–2.5 m M ; AMP: 0.5–1.5 m M ) under forward assay conditions (Fig. 6A,B). Dixon analyses of the inhibition of leishmania AK with Ap 5 A at saturating concentrations of the other substrate (ATP: 2.5 m M ;AMP 1.5 m M ), showed a competitive inhibitory pattern for both substrates, with K i values of 190 n M and 160 n M for ATP and AMP, respectively. Ap5A also inhibited growth of L. donovani promastigotes in vitro (Fig. 7) with IC 50 value of 27 ± 5 l M . The effect of Ap 5 A on leishmania growth couldbepartiallyreversedwithADP(IC 50 ,48± 9 l M ) but addition of ATP to the culture medium did not show any reversal (IC 50 ,33± 6). The reversal with ADP was statistically significant (P < 0.01%). Ap 5 A did not show a direct effect on luciferase activity of the transgenic Leish- mania promastigote extracts. Fig. 4. Heterologous expression of recombinant AK from L. donovani. E. coli cells (strain XL1-Blue) transformed with the plasmid PQE30- AK were grown and the cultures were induced with 0.1 M IPTG for 5 h. Samples of soluble and insoluble fractions were loaded onto SDS/ 12% polyacrylamide gels under reducing conditions. (A) Lane 1 solu- ble fraction; lane 2 insoluble fractions. Lane 3 shows the inclusion bodies after purification as outlined in Materials and methods. The arrow shows the 26-kDa expression product. (B) Forty micrograms exponential phase (day 3) promastigotes were loaded onto SDS/12% PAGE. The separated proteins were transblotted onto nylon mem- branes and probed with an anti-AK polyclonal antibody. MWM, molecular mass markers; CNT, preimmune serum. Fig. 5. Half-life of recombinant L. donovani AK at different tempera- tures. Inclusion bodies from E. coli XL1-Blue, transformed with PQE- 30-AK, were washed, folded and purified as described in Materials and methods. Purified enzyme aliquots were maintained at 4 °C(m), 13 °C (j) and 26.5 °C(d) and AK activity was measured at different time points. Each point represents the mean of two different experiments. Fig. 6. Dixon plots of the inhibition of recombinant AK by the specific inhibitor Ap 5 A. Six micrograms recombinant L. donovani AK were incubated in presence of several concentrations of ATP (A) or AMP (B) and different concentrations of the inhibitor. Each point is the mean of three separate trials. 4344 H. Villa et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Discussion Amino acid sequence of AK2 cloned from L. donovani shows significant homology with the corresponding enzyme from S. cerevisiae (28%) [17], Homo sapiens (29%) [16], P. falciparum (15%) (AF308612), T. vaginalis (25%) [18] and T. brucei rhodesiense (14%) (AF047722). Analysis of leishmania AK sequence by BLAST and CD search revealed the presence of a large (38 amino acids) LID domain, which confirms its identity as a type 2 AK. A threonine residue specific to AMP binding is present at position 31, and the P-loop attributed to the ATP binding site is mostly conserved, although residues Q10 and G11 seem to be translocated. Within the AMP binding domain, some amino acid residues are well conserved (L59, F87, R89) while two others, a leucine and a valine, are replaced by V35 and I60, respectively. A Mg 2+ binding site, generally consisting of two aspartic acid residues, contains two glutamic acid residues at positions 33 and 85 in AK2 of L. donovani.SomeAKsarealso known to bind a Zn 2+ cation, which plays a role in protein stabilization and folding, although it is not essential for catalysis. The well-established C-X 2 -C-X n -C- X 2 -C motif that provides a Zn binding-site in some AKs (i.e. Chlamydia pneumoniae [27] or P. falciparum AF308612) is absent here, suggesting that L. donovani AK uses only Mg 2+ as a metal cofactor. Three different views of a theoretical three-dimensional ribbon model diagram of the leishmania AK2, which were obtained using the SWISS-MODEL (Automated-Knowledge-Based- Protein-Modelling-Server; http://www.expasy.ch) (Fig. 8), shows the structure of leishmania AK2 to be similar to Fig. 7. Effect of Ap5A on the growth of L. donovani promastigotes in vitro. Effect of Ap5A at varying concentrations (5–250 l M )was tested alone (control) and also in combination with ATP or ADP (1 m M ). Each point represents the mean ± SD of at least triplicate observations. Fig. 8. Three-dimensional ribbon diagram of L. donovani AK 2. The SWISS-MODEL (Automated-Knowledge-Based-Protein-Modelling-Ser- ver; http://www.expasy.ch) was used for prediction of theoretical three- dimensional structure of the enzyme. The three different views were obtained by simple rotation of the same model. The flexible LID domain appears at top of the three figures, NMP-binding site and ATP binding domain are displayed, NH 2 - and COOH-termini are also indicated. Ó FEBS 2003 L. donovani adenylate kinase (Eur. J. Biochem. 270) 4345 that of E. coli AK. The flexible LID domain which provides open and closed conformation to the protein during binding of the substrate is shown in the top-most portion of the protein. ATP may bind to the glycine loop on the left flap portion (Fig. 8B) and AMP may bind to the AMP flap shown on the right side (Fig. 7B). AK displays a closed configuration in the presence of bound nucleotides and an open arrangement when products are released to the medium, thus configuring a movable structure [28]. Reduction in the activity of recombinant AK2 during storage at different temperatures indicate inactivation of the proteins which seems to be faster at 26 °Cthanat4and13°C. Heterologous expression in competent E. coli strains shows that the kinetic behaviour of L. donovani recombin- ant AK2 is hyperbolic for both AMP and ATP in the forward reaction. Like most AKs, the enzyme was strongly inhibited by Ap 5 A, with K i values of the order of those found for other sources for both AMP and ATP [24,26,27]. Southern analysis of the L. donovani AK gene showed that it is present as a single-copy gene into the genome. During the progress of this work, six putative AK genes on chromo- somes 4, 21, 25, 34(2) and 36 have been identified in the L. major genome (http://www.genedb.org/genedb/leish/ index.jsp). The L. major AK gene CHR34_tmp.344, cor- responds to the L. donovani AK2 gene described in this paper. The amino acid sequence of L. donovani AK2 gene shows 96% homology to the CHR_tmp.344 while homo- logy in nucleotide sequence was found to be 94%. Other putative AK genes were significantly different. The results of Southern blotting also indicated the AK2 as a single copy gene in L. donovani. Investigation of transcription of the AK gene by Northern blot analysis during the Leishmania life cycle detects a single mRNA transcript of  1.9 kb. Leishmania cell seem to have strong regulation for expres- sion of AK2 gene as the relative abundance of AK mRNA during the early logarithmic phase was much higher than in other phases of the promastigote cell cycle. AK has been detected in glycosomes in Leishmania [10]. Recently, three new putative AK gene sequences, one of them is incomplete (AL 139794), have been identified by the L. major Friedlin Genome Project. One of the L. major AK genes seems to encode for a short AK isoform (AQ 852692), with an LID domain of 18 amino acids, while the other two genes (AL354533) encode longer AKs. Moreover, the AL354533 and AQ 852692 isoforms lack the threonine 31 residue, which is replaced by a valine or a serine, respectively. This suggests possible involvement of AKs also in CMP/UMP metabolism in leishmania. The recombinant leishmanial AK2wasalsoselectivelyrecognizedbytheserumof hamsters infected with L. donovani (data not shown). AK2 may be a potential target antigen for a vaccine or diagnosis of leishmaniasis. The sequence of the AK genes identified in the leishmania genome, including the present one, differ significantly from each other. Diverse AK isoforms may be localized in different cellular compartments in leishmania and may have important roles in energy metabolism and adenine nucleotide equilibrium. Inhibition of leishmania growth with Ap 5 A, an inhibitor of leishmanial AK2 and its partial reversal by ADP indicate the importance of this enzyme in leishmania growth and proliferation. The effect of Ap 5 A on other metabolic functions of leishmania cell may not be ruled out at this stage. Detailed investigations of the distinct molecular characteristics of leishmania AK2 in comparison with its mammalian counterparts and its absolute need of the enzyme for growth and survival of the parasite within host macrophages are required to allow the development of selective inhibitors of the parasite enzyme as potential antileishmanial agents. In leishmania promastigotes AK has been found to be associated with glycosomes [10,11]. Beside the role of AK in maintenance and regeneration of intracellular ATP levels as shown in other organisms [7] partial reversal of antileishmanial action of Ap 5 A by ADP indicate that the enzyme might also be important for regeneration of ADP in leishmania, which may be required for continuous synthesis of ATP through substrate level phophorylation. AK in conjuction with an adenylate translocator has been shown to maintain the metabolic interconnection between malaria parasite P. falci- parum and the host erythrocyte [29]. High levels of ATP are generated in the malaria parasite by AK during intra- erythrocytic growth, and this is translocated to the eryth- rocytes. AK may also play a role in the ATP regeneration system required for flagellar movements in leishmania promastigotes as shown earlier in the cilia of Paramecium caudatum [7] or may also be a virulence factor as shown in Pseudomonas [12]. AK may thus be a potential drug target in leishmania. Acknowledgements This work was supported in part by Comisio ´ n Interministerial de Ciencia y Tecnologı ´ a (CICYT, grants PM98/0036 and BMC2002 04107-C02-02) and Junta de Castilla y Leo ´ n (JCyL grants LE01/00B and LE54/03). We also thank to Dr J. Zhou for his advice in AK refolding experiments, Dr N. Fasel, I. Segura, P. Bastien, and F. Fierro for their technical support in PFGE. BLT is supported by Cooperative scientific agreement grant from Center for Disease Control, Atlanta USA (U50-CCU418839) and also by a USDA cooperative agreement no. 58-6408-2-0009. References 1. Noda, L.H. (1973) Adenylate kinase. In The Enzymes,3rdedn, Vol. 8, (Boyer, P.D., ed.), pp. 279–305. Academic Press, New York. 2. Yan, H. & Tsai, M.D. (1999) Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity. Adv. Enzymol. Relat. Areas Mol. Biol. 73, 103–134. 3. Bandlow, W., Strobel, G. & Schricker, R. 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Proteins 32, 276–288. 29. Kanaani, J. & Ginsberg, H. (1989) Metabolic interconnection between human malaria parasite Plasmodium falciprum and its host erythrocyte: regulation of ATP levels by means of an adenylate translocator and adenylate kinase. J. Biol. Chem. 264, 3194–3199. Ó FEBS 2003 L. donovani adenylate kinase (Eur. J. Biochem. 270) 4347 . Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani He ´ ctor Villa 1 , Yolanda Pe ´ rez-Pertejo 1 ,. molecules of ADP. This study describes cloning and functional characterization of the gene encoding AK2 from a genomiclibrary of Leishmania donovani and also its

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